1. Field of Invention
The present invention relates to a device or apparatus for gel electrophoresis, in particular, to such devices or apparatuses adapted for the safe handling and disposal of agarose-type gels containing dyeing compounds used therewith.
2. Prior Art
Gel electrophoresis, a commonly used method on molecular biology research, is a technique designed to separate, identify and purify DNA, RNA and protein molecules based on their weight, size and shape. This technique, which is simple and rapid to perform, is carried out by first preparing a gel. When the gel is ready it is placed in a gel box, immersed in a buffer solution, and connected to a power source. Once stimulated by the electric field that is set up in the gel, the molecules move through the gel matrix at different rates. The migration rate for each species of molecule is dependent upon the electrical charge, the size and shape of the molecules, as well as on the composition of the gel. Most commonly, the smaller molecules will move through the matrix at a quicker pace than those of a larger size. Sufficient quantity of buffer (typically TAE, TBE or protein running buffer) is generally used to ensure that the electric field is set up in the gel, and that the gel is covered with it and thus prevent the gel from drying out during electrophoresis. When loading a sample containing the molecule species of interest into the gel, a loading dye is typically used. The loading dye normally allows easy visualization of the solution during the loading process, as well as enabling the density of the sample to be increased to ensure that the sample is fully and evenly accommodated in a corresponding well in the gel, and further allows visualisation of the migration during electrophoresis.
The most commonly used gels are prepared with either agarose or acrylamide, either one of which can be provided in varying shapes, sizes and thicknesses. The deciding factor as to which particular gel and its physical attributes is generally related to the size of molecule being separated and the desired experiment to be performed by the user.
Acrylamide is usually chosen for relatively small molecules such as proteins, while agarose is used for larger molecules such as DNA or RNA, and agarose is the preferred choice for horizontal gel electrophoresis, typically cast in open trays by users.
Typically, it is desirable to visualize and to document the results of the electrophoresis separation test. In electrophoresis separation of DNA or RNA molecules, this may be accomplished by immersing the gel slab after the electrophoretic separation has been completed in a solution of a fluorescent compound which emits visible light when exposed to a ultraviolet (UV) light. Other methods of staining the gel are also known, for example by adding a suitable dye to the gel while casting the same. A widely used fluorescent compound is ethidium bromide. However, many types of such fluorescent compounds, including ethidium bromide, Acrydine Orange, SYBR Green I, and SYBR Green II, as well as acrylamide and also some components used together with some agarose gels, are toxic and/or carcinogenic, and contact with users must be strictly avoided, particularly when disposing of the gels after use.
It is therefore an aim of the present invention to provide a device and method which overcomes the limitations of prior art electrophoresis devices and methods.
It is another aim of the present invention to provide a device for enabling safe handling and disposal of gels which may contain harmful substances.
It is another aim of the present invention to provide such a device that is simple to use.
It is another aim of the present invention to provide such a device that is relatively simple mechanically and thus economic to produce.
It is another aim of the present invention to provide such a device that is adapted for use with regular horizontal electrophoresis equipment.
These and other aims are accomplished in the present invention by providing a precast cassette for horizontal electrophoresis, in particular a disposable and closed cassette for horizontal electrophoresis. In particular, the cassette comprises preferably a pair of traps, one at each longitudinal end of the cassette, each trap comprising an absorption material for preventing toxic material from migrating out of the cassette. This is an important safety feature, particularly in view of the handling of the horizontal electrophoresis apparatus and of the cassette during use thereof, and more so in view of the disposability of the cassette, which thus minimizes the risk of human contact with toxic substances comprised in the cassette.
In the preferred embodiment, the cassette comprises a box-like construction, having a bottom flat base and four vertical walls joined thereto about its periphery, and an upper cover mountable onto the vertical walls to define a gel chamber into which gel may be precast. The cassette also comprises openings at two opposite ends of the bottom base to enable ionic communication between the gel and an electrolytic solution in which the cassette may be partially immersed. The openings are preferably comprised in downwardly extending hollow leg members running the transverse length of the cassette at two longitudinal ends thereof, the leg members comprising gel in ionic communication with the main body of gel within the cassette. This design is particularly adapted for using the cassette with standard ion exchange chambers. Activated carbon or the like is provided in the legs and also in a second trapping chamber to absorb dangerous materials such as the dyeing compounds.
U.S. Pat. No. 3,888,759 discloses a gel cassette having a substantially box-like construction, having a downwardly depending transversely extending hollow leg at each longitudinal end of the cassette. The device appears to be reusable, providing the user with different options, and it appears intended for the user to cast the gel each time, rather than providing a precast package. There is no disclosure or suggestion of a trap for toxic materials, and in fact teaches away from this concept.
U.S. Pat. No. 5,443,704 discloses a substantially box-like container assembly for packaging prefabricated gels, containing more than one precast gel in a stacked arrangement. No trap for harmful substances is disclosed therein.
U.S. Pat. No. 5,064,769 discloses a gel for immunoassay of a single protein species in which the horizontal gel comprises a first part made from acrylamide gel having a proportion of agarose (0.7%) and a second part made from agarose gel. No trap for harmful substances is disclosed therein.
In U.S. Pat. No. 3,930,983 an arrangement and process are disclosed for determining antigens, in which a support plate is coated with an agar or agarose as a matrix in successive gel strips. No trap for harmful substances is disclosed therein.
U.S. Pat. No. 5,582,702 is directed to a self-contained electrophoresis apparatus comprising a housing having a gel body accommodated therein together with ion exchange matrices and electrodes, which are electrically connectable to an external power source. The apparatus is thus not generally compatible with existing ion exchange chambers currently used for horizontal electrophoresis. While it is presented as optionally “disposable”, the apparatus is nonetheless complex, and does not appear inexpensive in comparison with simple precast gels, such as those described in U.S. Pat. No. 5,443,704, for example. Moreover, the apparatus contains elements which are not normally considered disposable, notably the electrodes and ion exchange matrices. In fact by being fully closed, in particular regarding the lower side thereof, the apparatus cannot be used with standard ion exchange apparatuses, and thus needs a dedicated stand having electrical connection points for the electrodes. Optionally, ethidium cations may be released into the gel by one of the ion exchange matrices within the housing, which may be simply disposed after use. However, and as stated earlier, the complexity of the apparatus renders this a rather expensive solution for the disposal of the contaminated gel. Furthermore, no traps are actually provided for retaining the contaminants therein—therefore, if any openings were to be made, for example, at the lower part of the cassette, for example as in the present invention, the contaminants could flow out, in contrast thereto.
EP No. 471949 discloses a capillary tube for performing capillary zone electrophoresis. The tube is modified by including a polystyrene frit that divides the tube into a downstream free zone, and an upstream zone which can comprise a polyacrylamide stacking gel. The gel plug functions as a filter to pre-treat the samples that are to be analyzed in the free zone of the tube. This is in contrast to the present invention, wherein the traps are placed downstream to treat the contaminants in the gel during and at the end of the electrophoretic process, and not prior to the beginning thereof. In fact, the samples in the present invention do not generally require pretreatment as described in this patent. Furthermore, the present invention uses agarose gel with an absorption material for retaining therein a target substance. On the other hand, in EP No. 471949 uses polyacrylamide, which is also toxic and use thereof would be detrimental in the present invention as a trapping gel, and in fact, counterproductive for this purpose. Thus, this publication teaches away from the present invention.
WO92/17259 describes a method for identifying a solute of interest in an effluent stream. A sample containing the mixture to be separated is passed through a first system capable of partitioning the components of the mixture, and a detector provides a first output that describes the temporal and/or spatial sequence of components exiting the first system. The effluent stream is then passed through a second system capable of extracting a solute of interest from the effluent, and a detector provides a second output that describes the temporal and/or spatial sequence of components exiting the second system, which no longer includes the solute of interest. The solute of interest can then be identified in the first output by comparing this to the second output. This method is thus directed at identifying a substance in a first separating system by employing a parallel second separating system, and is thus very different to the present invention, in which only a single electrophoresis process is employed, the target substance being removed during that process. WO 92/17259 does not address the problem of, nor does it provide a solution for, the trapping of toxic substances in an electrophoresis process, less so in the manner of the present invention.
WO 95/20155 relates to a sample holder in the form of a well, into which a sample and a first molten gel is introduced. When the first gel/sample mixture has solidified, the sample holder is applied against one end of a second gel slab, such as to bring the first gel/sample solidified mixture in ionic contact with the second gel. At no time is the first gel in solidified form brought into contact with the second gel prior to introducing the sample. WO 99/30145 relates to a slotted electrophoresis gel composition and methods for use, for providing a multilayered gel for vertical gel electrophoresis. It does not address, nor provide a solution for, the problem of forming stable sample wells for horizontal electrophoresis in an acrylamide gel. These publications do not address the problem of, nor do they provide a solution for, the trapping of toxic substances in an electrophoresis process, less so in the manner of the present invention
Other publications of background relevance to the present invention include WO 98/10277, U.S. Pat. No. 5,228,971, U.S. Pat. No. 5,827,418, U.S. Pat. No. 3,873,433, EP No. 971229, WO 95/14921, DE 3232685, EP No.199470 and U.S. Pat. No. 3,803,020.